Muscleblind-like 1 (MBNL1) is an alternative splicing factor whose improper sequestration is a primary component of the genetic disease myotonic dystrophy (DM). DM is caused by a toxic RNA that binds MBNL1 and alters its free cellular concentration leading to mis-splicing of MBNL1 targets. MBNL1 targets vary widely in their binding site composition, location, and affinity for MBNL1. We propose to modulate cellular MBNL1 protein concentrations to investigate the response of the entire MBNL1 splicing program under MBNL1 levels relevant to DM and tissue specific alternative splicing regimes. This novel approach will unveil the vast variation of regulation that occurs simply by changing the cellular concentration of one key alternative splicing component. With this data we will begin to understand the variety of ways MBNL1 can regulate alternative splicing at disparate targets. We will perform mechanistic studies using a few representative MBNL1 regulated events to address how the context of its target RNAs affect its mode of action. We will investigate how all MBNL1 splicing targets are altered within different patient samples based on the degree to which MBNL1 is sequestered to the toxic RNA. These studies will lend insight into how perturbing MBNL1's functional concentrations leads to a debilitating disease through alternative splicing mechanisms. Aim 1: Determine the effect of altering MBNL1 cellular concentrations on alternative splicing. Sub-Aim 1A. Determine the dose-response relationships between MBNL1 and splicing reporters using a tunable MBNL1 expression system. Sub-Aim 1B: Determine the contribution of MBNL1 binding events to dose-response behavior using in vitro binding, mutational analysis, and in vitro splicing assays. Aim 2: Investigate MBNL1 sensitivities of endogenous splice targets. Sub-Aim 2A: Determine MBNL1 dose responses of endogenous splice targets. Sub-Aim 2B: Determine MBNL1 sensitivities of splicing events in DM patient samples with different CTG repeat lengths.